Human apolipoprotein A-I (apo A-I) is the major protein constituent of high density lipoproteins (HDL) and lymph chylomicrons. The liver and small intestine are the primary sites of synthesis of apo A-I. In these organs, apo A-I is synthesized as a precursor protein (preproapo A-I). Cotranslational cleavage of the prepeptide occurs intracellularly and proapo A-I is secreted into the plasma and lymph.
Proapo A-I has six additional amino acids (Arg-His-Phe-Trp-Gln-Gln) attached at the amino terminal end of apo A-I. Upon reaching the vascular space, proapo A-I is cleaved, in vivo, by a specific proteolytic enzyme (apo A-I propeptidase) to yield mature apo A-I.
Mature apo A-I is a single unglycosylated polypeptide of known sequence composed of 243 amino acid residues (H. B. BREWER et al., Biochem. Biophys. Res. Commun. 80, (1978), 623-630); it serves as a cofactor for the plasma enzyme lecithin: cholesterol acyltransferase, which is responsible for the formation of most cholesterol esters in plasma. Defects in apolipoprotein structures or biosynthesis may result in disorders of the plasma lipid transport system and in the development of coronary artery disease. Low levels of apo A-I and HDL in plasma has been shown to be a strong risk factor for heart attacks (myocardial infarction) and other atherosclerotic vascular diseases. Mutations in the gene coding for apo A-I namely have been associated with reduced HDL levels and with premature coronary artery diseases.
Apo A-I and HDL are the major plasma components that participate in the transport of cholesterol from peripheral tissues (arteries) to the liver (so called reverse cholesterol transport) for excretion from the body. Since accumulation of cholesterol in the arteries is the hall-mark and most important process of atherosclerosis, stimulation of reverse cholesterol transport by supplying apo A-I may retard and reverse the atherosclerotic process and hence diminish the incidence of heart attacks.
Maturation of proapo A-I into apo A-I can occur quantitatively and extracellularly with a residence time in the blood of less than 12 hours. Since proapo A-I is the major, if not the sole, precursor to the mature apo A-I, it could be used in replacement therapy whenever HDL levels decrease, for instance in hereditary or acquired deficiencies. Because of the therapeutic utility of apo A-I in general, researchers have been looking for techniques to produce apo A-I in large quantities. Conventionnaly, such techniques have involved purification of apo A-I from blood plasma. Several publications (P. CHEUNG and L. CHAN, Nucleic Acids Res. 11, (1983), 3703-3715; J. J. SEILHAMER et al., DNA, 3, (1984), 309-317 and Japanese patent application No. 96998/86) have shown that the complementary DNA coding for preproapo A-I can be obtained by well-known genetic engineering techniques. R. LORENZETTI et al., (FEBS Lett. 194, (1986), 343-346) have shown that apo A-I can be expressed in E. coli as an uncleavable fused protein with .beta.-galactosidase. Attempts to express mature apo A-I as an unfused protein were unsuccessful, however.
In the above-mentioned Japanese patent application No. 96998/86, there is described the expression of a human apoliprotein A-I-like protein in E. coli, which have been transformed with a plasmid pHAIE-I containing the structural apo A-I gene under control of the tac promoter. The structural gene is not complete, however, and consists of the condons for amino acids +4 to +243, preceded by an ATG translation initiation codon. As a consequence, the expression product obtained contains N-terminal methionine (corresponding to the ATG codon) which might cause secondary effects when used in therapy. J. B. MALLORY et al. (J. Biol. Chem. 262, (1987), 4241-4247; PCT International patent applications WO 86/04920 and WO 87/02062) disclose expression of human apolipoprotein A-I in Chinese hamster ovary cells (animal cell culture). The construct used contains the entire human preproapolipoprotein A-I gene. The Chinese hamster ovary cells seem to process the proapo form to the mature apo form, since only 5-10% of the secreted apo A-I protein is proapo A-I, the remainder being the mature apo A-I protein. The productivity of the system is relatively low, since 0,55.times.10.sup.6 cells secrete only 25-30 .mu.g/ml of apo A-I in a 24-hour period. In the PCT International patent application WO 87/02062, some examples are concerned with expression of human apolipoprotein A-I in other hosts, such as E. coli (bacterial host) and S. cerevisiae (yeast). None of the constructs used contains the genetic information for the proapo form, however, and the resulting protein is not human proapolipoprotein A-I.